Project Summary This project is focused on developing a novel in vitro platform for studying the impact of the enteric nervous system on epithelial phenotype. There is a need for a simplified model of the human gut-enteric axis, as a clear connection exists between gut and neural health and dysfunction, but the underlying regulatory mechanisms are not well understood. The enteric nervous system is known to have tremendous impact on gut homeostasis, especially the potential for inducing an anti-inflammatory response with vagus nerve stimulation. However, due to the non-specificity of bioelectric vagal targeting and the limitations of probing innervated organs in vivo, clinically relevant stimulation regimes for the gut have yet to be identified. Human intestinal epithelial cells express receptors that are specific for enteric neurotransmitters, such as acetylcholine, which may be activated during electrical stimulation leading to an anti-inflammatory phenotype. Thus, a microphysiological system that recapitulates key components of the human gut-enteric-axis, including shear flow, oxygen saturation, bioelectric stimulation, primary human epithelium, and primary human enteric neurons would be a valuable tool for advancing scientific discovery, healthcare, compound screening, and biomedical research. Current organ-chips generally utilize specialized equipment and microfabrication techniques for platform development, limiting dissemination, as well as do not include primary human small intestinal epithelium or enteric neurons. The approach here describes the development of a laser-fabricated, cut and assembled body-chip for a humanized gut-enteric axis (hGEA). The team has worked together to establish a prototype hGEA and establish primary enteric and epithelial cultures, and thus have demonstrated their complimentary teaming ability towards product development. Two parallel and synergistic aims will be pursued, with platform development in Aim 1 utilizing laser machining and assembly to fabricate the hGEA with microelectrode array electrophysiology capabilities, followed by characterization of neural and epithelial responses on chip compared to static transwell controls over 0-14 days, and lastly oxygen and shear flow modeling to recapitulate physiological conditions. Aim 2 will investigate the impact of electrical stimulation of enteric neurons to modulate a chemically induced inflammatory phenotype in the primary human epithelium, and characterize especially (but not limited to) nicotinic acetylcholine receptor proteins, trans epithelial electrical resistance (TEER), enzyme and mucus production, and cytokine release as markers of epithelial health. Experiments will be benchmarked to conventional Caco-2 models and static controls. The successful completion of the first ever in-vitro human GEA will accelerate the mechanistic study of gut disease, including inflammatory disorders, and advance therapeutic target discovery by enabling analysis on an accessible and cost-effective, laser cut and assembled microphysiological platform.